Polyketides are an important class of natural products responsible for the development of many human therapeutic, veterinary, and agricultural products (e.g. FK506, lovastatin, and avermectin). The enzymes which synthesize these compounds, polyketide synthases (PKSs), have been the target of various molecular engineering methods aimed at producing either improved analogs of existing pharmaceuticals or combinatorial libraries of novel polyketides. Modular PKSs—such as the 6-deoxyerythronolide B synthase (DEBS) shown in FIG. 1 have been altered by such techniques to produce new polyketide structures derived by genetic manipulation of one or more of the enzymatic domains contained in such PKS enzymes (see U.S. Pat. No. 5,962,290 and PCT Pub. No. 98/49315, supra).
These first-generation successes have since led to a rapid proliferation of genetically engineered PKSs that produce novel polyketides or ‘unnatural’ natural products (see PCT Pub. Nos. 99/61599, 00/024907, and 00/026349, each of which is incorporated herein by reference). Recent work has culminated in the generation of libraries of ˜100 macrocyclic compounds, illustrating the potential to create libraries with significant complexity and diversity (see PCT Pub. Nos. 00/063361 and 00/024907, each of which is incorporated herein by reference).
The ability to manipulate predictably the catalytic activities of these multifunctional enzymes represents significant technological achievements in protein engineering. However, one of the current challenges to the construction of very large compound libraries (>1000 compounds) is the decline in production levels associated with many genetically modified PKSs, particularly those in which multiple domains have been modified (PCT Pub. Nos. 00/063361 and 00/024907, supra). While it is desirable to use PKS structure-activity knowledge to help guide more optimal engineering of PKSs, due to the complexity and size of these enzymes, the current understanding is relatively limited, and progress has been slow. It is therefore important to develop complementary approaches that do not rely on a detailed understanding of the enzymatic architecture to improve combinatorial biosynthesis technologies.
One possibility leverages the significant resources that are generally devoted over the course of many years towards establishing commercial processes to produce natural product pharmaceuticals. Almost inevitably, extensive strain improvement and process development programs are undertaken to increase the yield of compound from the natural producing organism, often achieving greater than 100-fold increases in titers. A number of microorganisms have been optimized through random mutagenesis for bulk production of highly valuable compounds, including penicillins, macrolide antibiotics, and lovastatins. Although this conventional approach to strain improvement could be applied to strains carrying engineered PKSs, it is a labor-intensive process, especially given the potentially large numbers of mutant strains that could be generated by combinatorial biosynthesis. However, if overproduction capabilities of existing industrial strains could be applied to increase titers of polyketides derived from engineered PKSs, it would present an attractive and economical solution. Not only would overproduction increase the accessibility of compounds produced at levels currently too low for screening, but also the size of libraries created could be enlarged, because more modifications could be introduced into a PKS before productions levels became too low. The broad applicability of such strains, however, depends on what factors are responsible for overproduction, and whether production from a recombinant PKS would be increased in an overproduction background.
The present invention meets the need for a generic host cell capable of producing a polyketide at levels significantly higher than in other host cells.